Cholesterol, a component of all eukaryotic plasma membranes, is essential for the growth and viability of cells in higher organisms. However, high serum levels of cholesterol cause disease and death by contributing to the formation of atherosclerotic plaques in arteries throughout the body. The major site of cholesterol synthesis in mammals is the liver. Appreciable amounts of cholesterol are also formed by the intestine. The rate of cholesterol formation by these organs is highly responsive to the amount of cholesterol absorbed from dietary sources. Cells outside of the liver and intestine acquire cholesterol from the plasma rather than by synthesising it de novo. Cholesterol and other lipids are transported in body fluids by lipoproteins, which are classified according to increasing density. A lipoprotein is a particle consisting of a core of hydrophobic lipids surrounded by a shell of polar lipids and apoproteins. These lipoproteins have two roles: they solubilize highly hydrophobic lipids and they contain signals that regulate the movement of particular lipids in and out of specific target cells and tissues. Cholesterol is transported in body fluids by low-density lipoproteins (LDL) which binds to a specific receptor on the plasma membrane of non hepatic cells. The receptor-LDL complex is then internalised into the cells by a transport mechanism known as receptor mediated endocytosis (Goldstein et al. 1979). The low density lipoprotein (LDL) receptor is the prototype of a family of structurally related cell surface receptors that mediate endocytosis of multiple ligands in mammalian cells.
The LDL receptor consists of 822 amino acid residues and exhibits a molecular weight of 164000. It is composed of several domains some of which share sequence homology with other proteins. Its NH2-terminal ligand-binding domain consists of 292 residues, arranged in 7 cysteine-rich imperfect repeats. Each repeat contains six cysteine residues which are disulphide bonded in the pattern one to three, two to five, and four to six. (Bieri et al. 1995). This domain is followed by four additional domains: the first consists of 400 amino acid residues and is homologous to the EGF receptor, the second consists of 58 amino acid residues rich in O-linked sugars, the third is a single trans-membrane domain of 22 amino acid residues and the fourth is a cytoplasmic domain of 50 amino acid residues (Sudhof et al. 1985), (Brown et al. 1986).
The physiologic importance of the LDL receptor was revealed by Brown and Goldstein's studies on familial hypercholesterolemia (FH). The disease was found to be due to a molecular genetic defect resulting in the absence or deficiency of functional receptors for LDL (Brown et al. 1976). Several classes of FH mutations have been characterised. (Goldstein et al. 1975).
A soluble form of the sLDLR exhibiting antiviral activity was identified and isolated from the culture supernatant of interferon-induced cells (Fischer et al. 1993) and in body fluids (Fischer et al. 1994). Several interferon-induced proteins have been identified that are instrumental in the induction of the antiviral state by IFNs. One such protein exhibiting antiviral activity was produced and accumulated in the culture supernatant of human amnion WISH cells. This protein was purified to homogeneity and identified as the sLDLR (see EP 0 553 667 and Fischer et al. 1993). The sLDLR was found to be secreted into the medium by mammalian cells that enter an antiviral state in response to interferon. In contrast to interferon, sLDLR does not induce an antiviral state in the cells but is antiviral by itself. It was found that sLDLR apparently has to be present throughout the process of viral replication maturation and budding suggesting it might be involved in a complex process that leads to the inhibition of virus assembly or budding (unpublished data). Endocytosis of the hepatitis C virus has been recently shown to be mediated by LDL receptors on cultured cells (Agnello et al. 1999). These and other findings suggest that the family of LDL receptors may serve as viral receptors. Therefore, antibodies rised against the sLDLR receptor may block the entry and budding of viral particles by binding to the cellular LDL receptor.
The only available monoclonal antibody to LDLR known so far is C7, an antibody to bovine LDLR (Beisiegel et al. 1981, commercially available from Amersham, UK) which was prepared by immunization of mice with the bovine adrenal cortex LDLR purified to homogeneity. Membranes from the bovine adrenal cortex were solubilized and the receptor was partially purified by elution from a DEAE-cellulose column (Beisiegel et al. 1981). The antibody to the bovine LDLR only weakly cross-reacts with human LDLR.
In fact, the C7 Mab to bovine LDLR was found to have significant disadvantages when used for detection and quantitation of recombinant human LDLR:    a) It has very low affinity to the human LDLR    b) It significantly cross reacts with cell culture derived impurities
Specific antibodies to human LDLR were not previously available. This appears surprising since it is very common to raise antibodies against novel proteins, be it for purification, identification or for assay development purposes. It is possible that such antibodies have not been generated so far, since a condition for generating monoclonal antibodies is the availability of sufficiently large amounts of highly purified antigen which allow efficient immunization of mice. A highly purified antigen is one which appears as a single major peak in RP-HPLC. Furthermore methods for identification and quantitation of the antigen during purification processes were not easy to establish. In accordance with the invention, the antiviral activity assay described herein was employed for the identification of LDLR during purification processes.
There exists a need to generate specific Mabs to human soluble LDLR to provide the means for developing an efficient immunoassay (ELISA) and for the identification of the protein in Western blot. These antibodies are required for the monitoring and quantitation of the recombinant human soluble LDLR during development of the production and purification processes of the recombinant protein and for detection of the natural protein.